Neuregulins for prevention and treatment of damage from acute assault on vascular and neuronal tissue and as regulators of neuronal stem cell migration

ABSTRACT

Neuregulin, a known neuroprotein, has been found to ameliorate or prevent damage caused by mechanical or chemical assault to blood vessels and, when administered into the cerebral spinal fluid, can ameliorate damage to neuronal tissue caused by stroke, inflammation or organophosphate neurotoxins. Additionally, neuregulin has been found to be useful for enhancement of stem cell migration from the ventricle to the site of injury to the brain.

This application claims priority from Provisional Patent Application60/713,681 filed Sep. 2, 2005.

This invention was partially supported by the United States GovernmentNIH grant NS34194 and an NSF Center for Behavioral NeuroscienceCooperative Agreement (#IBN-9876754). Hence, the United StatesGovernment has certain rights in this invention.

BACKGROUND AND FIELD OF THE INVENTION

This specification discloses novel means for treatment of acute vascularconditions which may arise from mechanical or chemical damage to thevascular system or that may arise from sudden decrease in blood supplyto the brain, as occurs in obstructive stroke or damage to neuronaltissue that arises from neurotoxins by appropriate administration ofneuregulin in accord with the teachings herein.

Neuregulins are a family of multipotent growth factors that includesacetylcholine receptor inducing activities (ARJAs), growth factors,heregulins, and neu differentiation factors. Neuregulins' effects appearto be mediated by interactionwith a class of tyrosine kinase receptorsrelated to the epidermal growth factor receptor. Neuregulins stimulatethe tyrosine phosphorylation of these receptors and the subsequentactivation of various signal transduction mechanisms. Neuregulins aresynthesized as transmembrane precursors consisting of either animmunoglobuline-like or cysteine-rich domain, and EGF-like domain atransmembrane domain and a cytoplastic tail. The EGF-like domain ofNRG-1 appears to be sufficient for activation.

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells and itsreceptors are localized in the underlying smooth muscle cells. However,its use in treatment of acute conditions and for repair of damagedtissue by enhancing migration of stem cells to damaged areas to providenew neuronal tissue has not previously been disclosed.

Atherosclerosis is a major cause of death in Western civilizations,leading to both heart attack and strokes. Atherosclerosis is a complex,chronic inflammatory disease of the arterial vessel wall which involvesmultiple processes including endothelial dysfunction, inflammation,vascular smooth muscle cell (VSMC) proliferation, and matrix alteration.Damage to the endothelial lining of the arterial wall due toangioplasty, insertions of stents or catheters and atherosclerosis allinduce the release of pro-inflammatory cytokines and growth factors thatstimulate normally quiescent VSMC to migrate and proliferate. VSMCproliferate and migrate from the medial layer of the vessel into theintima resulting in neointimal hyperplasia, which is also a major causeof restenosis after angioplasty or cardiac surgery, especially surgeryon the heart valves. Mitogens, such as platelet derived growth factor(PDGF), are potent stimulators of VSMC proliferation and differentiationfollowing vascular injury. PDGF is produced by platelets, endothelialcells, smooth muscle cells and macrophages that infiltrate the artery inresponse injury and the release of PDGF afterlinjury contributessignificantly to the formation of the neointima.

Ischemic stroke occurs when the blood supply to the brain is obstructed.The neuronal death that ensues results from the induction of genesassociated with a number of additional cellular functions. A substantialbody of research implicates inflammation as a contributor to strokemorbidity. Ischemic stroke initiates an inflammatory response in theinjured brain and progresses for days after the onset of symptoms. Thereis evidence that inflammatory reactions are involved in the delayedischemic injury and result in poor prognosis of neurological outcome.

In response to cerebral ischemia, inflammatory cytokines, such as tumornecrosis factor (TNFα) and interleukin-1β (IL-1β), are induced in theischemic brain of animal models. Following ischemic injury, IL-1β andTNFα, have been shown to facilitate neuronal damage. These cytokinesinduce the expression of adhesion molecules, downstream pro-inflammatorymolecules and stress genes.

Chemical cause of neuronal damage include neurotoxins such as those usedin chemical warfare and in some pesticides. Recent studies havedemonstrated the existence of neuronal injury secondary to thestimulation of cholinergic pathways, which is associated withpro-inflammatory processes in the CNS following exposure toOrganophosphorus (OP). OP nerve agents are toxic chemicals that havebeen used by terrorists in military combat and against civilianpopulations. Current post-exposure medical counter-measures againstnerve agents (e.g. atropine, oximes and benzodiazepines) are useful inpreventing mortality, but are not sufficiently effective in protectingthe CNS from seizures and permanent injury. Therefore, new and moreeffective medical countermeasures to avoid post-exposure damage toneuronal tissues are needed. Recent studies have demonstrated theexistence of neuronal injury, secondary to the stimulation ofcholinergic pathways, which is associated with pro-inflammatoryprocesses in the CNS following exposure to OP nerve agents.

The neuregulins have been known to be involved in the survival andfunction of neuronal cells. A recent study using NRG-1 demonstrated theneuregulin blocked delayed neuronal death following focal ischemicstroke. However, the mechanisms that underlie the neuroprotectiveeffects of NRG-1 are unclear. Neurogenesis has been described in theadult mammalian CNS in the subventricular zone (SVZ) and dentate gyrusof the hippocampus. Studies have shown that neurogenesis may bestimulated from multiple cell types in the SVZ. Four cell types arefound in the SVZ: neuroblasts (type A), SVZ astrocytes (type B), rapidlydividing precursors (type C), and multiciliated ependymal cells. Eachtype is capable of giving rise to neurons and glia.

Stem cells research has attracted widespread attention and controversyover the past several years. Stem cells are undifferentiated, primitivecells with the ability to differentiate into various kinds of cells.Stem cells can be used to restore or regenerate tissue, which could beuseful in treating injuries or disease. Stem cell research iscontroversial because the best source of true pluripotent stem cells ishuman fetal tissue, which is harvested from destroyed embryos. Unlikeembryonic stem cells, adult stem cells are unspecialized,undifferentiated cells that exist in very small numbers amongspecialized cells in an adult organ or tissue. Their main function is tomaintain and periodically repair the tissues in which they are found.Adult stem cells are found in a number of locations, including thebrain, the bone marrow, peripheral blood, blood vessels, skeletalmuscle, skin, and liver. There is no controversy regarding the use ofhuman adult stem cells in research, since they can be retrieved from theindividual requiring the therapy.

Neurogenesis in the adult brain normally also occurs in two regions ofthe adult mammalian brain; the olfactory bulb and the dentate gyrus ofthe hippocampus. The neural stem cells (NSCs) destined for the olfactorybulb originate from the subventricular zone (SVZ), which lies along thelength of the lateral ventricle. In the developing CNS, NSCs comprise aself-renewing cell population able to generate neurons, astrocytes andoligodendrocytes. The newly generated olfactory bulb NSCs proliferateand migrate in the SVZ along the rostral migratory stream (RMS) towardsthe olfactory bulb. These NSCs differentiate into two kinds ofinhibitory interneurons in the olfactory bulb. Four ceil types are foundin the SVZ: neuroblasts (type A), SVZ astrocytes (type B), rapidlydividing precursors (type C), and multiciliated ependymal cells (EC)that line the ventricles. Focal clusters of rapidly dividing type Ccells are found scattered along the RMS. The stem cells in the adult SVZhave been shown to be SVZ astrocytes in one study. SVZ astrocytes divideto give rise to rapidly dividing immature precursors (type C) that inturn generate the neurons that migrate to the olfactory bulb anddifferentiate into neuroblasts (type A). However, other reports suggestthat EC may also serve as adult SVZ stem cells. New neurons in thehippocampus are derived from NSCs in the subgranular zone (SGZ) and giverise to granule cells that project to the CA3 region of the hippocampus.It has also been demonstrated that neurogenesis can occur in regions ofthe adult mammalian brain, like the neocortex, where it does notnormally occur, via manipulation of endogenous multipotent precursors insitu.

NSCs of CNS are patterned in vivo to generate neurons, oligodendrocytes,and astrocytes. In vitro, pluripotent NSCs generate lineage-restricted,self-renewing neuron-restricted progenitors (NRPs), and glial-restrictedprogenitors (GRPs), which subsequently develop into fully differentiatedneuron and glial cells, respectively. NRPs are mitotically active andelectrically immature, and they express only a subset of neuronalmarkers. NRPs undergo additional changes to develop into mature,functional neurons, NSCs, NRPs, and GRPs have been previously isolatedfrom mouse neural tubes that undergo self-renewal in defined medium, anddifferentiate into multiple neural phenotypes in mass culture. Whenisolated neuroepithelial NSCs are maintained in culture in the absenceof a substrate that supports adhesion, cells form neurospheres.Neurospheres contain a relatively homogeneous population of NSCs thatundergo self-renewal in response to either bFGF or EGF. It was observed,however, that after mitogen withdrawal, the NSCs were unable to undergoneuronal differentiation directly. These cells emerged from theneurosphere as NRPB. These NRPs were required to go through one or morerounds of cell division before neurogenesis could proceed to generate ofneurons.

SUMMARY OF THE INVENTION

It is the purpose of this invention to provide methods and compositionsof neuregulins for use in treatment of persons who have sufferedimpairment of neuronal function due to destruction of neuronal cells byincreasing migration of stem cells from the ventricle to the damagedareas of the brain. Intracerebroventricular administration of neuregulinresults in the mobilization and migration of endogenous NSCs/wv/vo.Results were shown when asubpopulation of these cells were studied whenlabeled with neuronal markers. Hence, neuregulins may be used asstimulators of adult neurogenesis and can be useful in treatingneurodegenerative disorders, including stroke. The NSC-derived cellsgenerated by NRG-1 are capable of repopulating regions of cell deathfollowing ischemic stroke. These findings may implicate a role for NRG-1in neuronal regeneration following ischemic stroke, resulting in anincrease in functional recovery following stroke.

It is a further purpose of this invention to provide methods andcompositions for treatment of the acute phase (0-72 hours) ofobstructive stroke by administration of neuregulin in conjunction withother treatment modalities such as glutamate receptor inhibitors whichblock the excitotoxic events of ischemia or t-PA, a clot disruptingagent, in order to decrease damage to neuronal tissue and injury arisingbecause of reperfusion after administration of agents such as t-PA. Theamelioration of neuronal damage is accomplished by administration of ainflammation inhibiting effective amount of neuregulin, in conjunctionwith the glutamate receptor inhibitor or a clot disrupting agent to apatient who has suffered an occlusive stroke, wherein the neuregulin isadministered within 72 hours of the onset of said occlusive stroke.

Administration via the carotid artery within the treatment time windowof up to 72 hours (therapeutic window) has not been disclosedpreviously. In order to access a particular portion of brain tissue inneed of exposure, neuregulins can be administered to a particular areaof tissue via fluoroscopy guided catheter in the usual manner used forcatheter-based therapy. Neuregulin may also be administeredintravenously in conjunction with reperfusion therapy followingocclusion of coronary arteries.

It is a further purpose of this invention to provide protection frompermanent neuronal damage after exposure to chemical damage such as thatresulting from exposure to nerve poisons such as organophosphates.

It is a further purpose of this invention to provide protection frompermanent damage to blood vessels from restenosis and artherosclerosisarising from physical assault such as placement of a balloon or stent inthe artery or diagnostic procedures such as cardiac catheterization.Furthermore, restenosis may develop after cardiac surgery, especiallysurgery on the heart valves. In addition to administration of neuregulinby intravenous route during or after the damaging occasion, a stent orcatheter for use in an invasive procedure may be coated with neuregulin.

In view of the above, the methods taught herein provide means ofpreventing damage from an acute assault on the neuronal and vasculartissue by appropriate, early treatment using neuregulins. Furthertreatment with neuregulins after damage has occurred may be achievedafter the inflammatory phase following the acute onslaught byintrathecal administration of neuregulin to enhance proliferation of newcells in the damaged areas of the brain by increasing migration of stemcells to the damaged regions of the brain.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the effects of neuregulin-1 onPDGF-stimulated VSMC proliferation. The open cycles represent controlcells. The filled cycles represent cells treated with neuregulin-1.

FIG. 2 is a diagram showing the effects of neuregulin-1 on NSCs isolatedfrom El 1 mouse telencephalon. The filled cycles represent controlcells. The open cycles represent cells treated with the EGF-like domainof neuregulin-1β (EGF-1 β).

DETAILED DISCLOSURE OF THE INVENTION

While it was previously shown that neuregulin-1 (NRG-1) increases theproliferation of neuronal restricted precursors (NRPs/type B cells) fromcultures of embryonic neural stem cells (NSCs/type C cells). It is nowfound that neuregulins are mitogenic to NRPs. Hence, endogenousneuregulins play important roles during CNS neurogenesis. However, aspecific role for neuregulins in the regulation of endogenousneurogenesis and its use in improving neuronal function by enhancingmigration of stem cells by intrathecal administration of neuregulin hasnot been disclosed previously.

While there have been previous suggestions that neuregulin might havetong term use in treatment of atherosclerotic conditions, its use inprevention or treatment of acute damage to the vascular system has notpreviously been disclosed. It has now been found that neuregulin alsoprevents mitogen-stimulated VSMC proliferation and migration. Hence, useof neuregulin represents a means of prevention of damage arising fromresponse to invasive procedures. To evaluate the potential role ofneuregulin as an agent for use in vascular injury, the effect of NRG-1on neointimal formation following balloon injury to the carotid arteryof the rat was examined. NRG-1 (2.5 ng/kg) was administered by tail-veininjection prior to injury and every two days following injury. Two weeksafter carotid artery injury, NRG-1-treated animals demonstrated a 50%reduction in lesion size compared to controls receiving the vehicle. Toexamine possible mechanisms for NRG-1 action, its effect on vascularsmooth muscle cell (VSMC) function was studied. A7r5 rat VSMC cultureswere pretreated with NRG-1 for 24 hours, and then stimulated withplatelet derived growth factor (PDGF) for 48 hours. NRG-1 significantlydecreased both baseline and PDGF-stimulated VSMC proliferation in adose-dependent manner. NRG-1 also blocked VSMC migration and preventedthe downregulation of α-smooth muscle actin by PDGF, indicating that itmay prevent VSMC phenotypic reversion following injury. These findingsdemonstrate NRG-1 as be a novel therapeutic agent for the treatment ofrestenosis and atherosclerosis.

Methods in Study of Prevention of Post-Trauma Damage to Blood Vessels:Experimental Injury, Harvest, and Tissue Preparation of Rat CarotidArteries

Male Sprague-Dawley rats (350-400 g) were balloon-injured using methodsas previously described in accordance with a protocol approved by theStanding Committee on Animals, Morehouse School of Medicine. Rats wereanesthetized with an intraperitoneal injection of xylazine (5 mg/kg bodyweight) and ketamine hydrochloride (90 mg/kg body weight). The leftcommon carotid artery was exposed by a 6-cm midline cervical incision.Proximal and distal blood flow was occluded by clamping. Polyethylene 10tubing was inserted retrogradely into the internal carotid artery andadvanced into the left common carotid artery. After gentle flushing ofthe artery with normal saline, the tubing was removed and a 2-French (F)Fogarty embolectomy balloon catheter was inserted. Balloon inflation to1.5 to 1.8 times the external diameter of the artery was achieved bycaliper measurement under stereomicroscopy. After holding the inflationfor 30 seconds, the catheter was removed. The uninjured right carotidartery was used as the control. Rats were treated with NRG-1β or NRG-1α(EGF-like domain, R&D Systems, Minneapolis, Minn. dissolved in 1%BSA/PBS) by tail-vein administration at a dose of 2.5 ng/kg body weight,starting at day 0 before injury, and continuing for every 2 days for thenext 14 days. Control rats were treated with vehicle (1% BSA/PBS). Theanimals were weighed before the procedure and at sacrifice to evaluatethe possible adverse effects of NRG-1. Vessels were harvested timepoints 0 and 14 days for mRNA analysis or histology. Injured vesselswere compared with their contralateral controls.

Tissue Processing and Quantitative Histomorphometric Analysis

Animals were euthanized with C0₂14 days after injury. Carotid arterieswere washed with saline to clear blood, embedded in Tissue-Tek OCTmedium and frozen using liquid nitrogen. Carotid sections were cut witha cryostat into cross sections of 12 μm taken from the center and distalportion of the vessels, and stained with hematoxylin and eosin. Themedial thickness was determined by the area of the internal elasticlamina subtracted from the external elastic lamina. Morphometry wasperformed using at least six individual sections of each arterialsegment and used to determine the lesion size expressed as intima/mediaratio. The intimal and medial layer thicknesses were measured using acomputer-based image analyzing program (Image J, NIH).

A7r5 VSMC Cultures

A7r5 rat aortic vascular smooth muscle cells (VSMC) (ATCC CRL-1444) wereobtained from American Tissue Type Culture (Manassas, Va.) and grown inDulbecco's modified Eagle medium supplemented with glutamine, 10% fetalcalf serum (FCS), and 1% Penicillin/Streptomycin at 37° C. in ahumidified incubator with 5% CO₂. Cells were passaged weekly. Allstudies were performed on cells from passages 9-12.

Determination of VSMC Proliferation

VSMC were seeded at a density of 1×1 0³ cells in triplicate wells of a96 well plate. After 24 hours, cells were serum starved in DMEM/F-12(Gibco; Carlsbad, Calif.) containing 0.1% FCS (low serum medium; LSM) toinduce quiescence. After 24 hours of serum deprivation, cells werepretreated with 0-200 nM of NRG-1α or NRG-1β for 24 hours. Cells werethen treated with 10 ng/mL of PDGF-BB for 48 hours to stimulate VSMCproliferation. For direct measure of cell number, cells were countedusing a Coulter counter. VSMC cell proliferation and viability was alsomeasured using the CellTiter 96 AQueous Non Radioactive CellProliferation Assay (Promega; Madison, Wis.) according to themanufacturer's protocol. After incubation at 37° C. in humidified 5% CO₂for 1 hour, the absorbance was recorded at 490 nm using a plate reader.Measurement of DNA synthesis was performed using the BrDU CellProliferation Assay (Calbiochem, San Diego, Calif.) according to themanufacturer's protocol.

Cell Migration Assay

Neuro Probe 48-well microchemotaxis chambers (Costar, Corning Inc.) withPVP-free polycarbonate filter (8.0 μm pore size) were used to measureVSMC migration. Quiescent cells were trypsinized and resuspended in LSMwith or without NRG-1 and incubated for 24 hours at 37° C. Cells werethen treated with PDGF which was added to the bottom well of the Boydenchamber and incubated for 48 hours at 37° C. Cells that migrated to thelower side of the filters were fixed and stained with the Diff Quickstaining kit (VWR Laboratory, West Chester, Pa.). The filters weremounted on glass slides and counted by light microscopy using ×100magnification.

Protein Purification and Western Analysis

Reactions were terminated by placing the cells on ice, aspirating themedium and adding ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 1 mMEDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, ImM sodium orthovanadate, 1mM phenyl methanesulfonyl fluoride, pH 8.0) for 30 minutes at 4° C.Harvested lysates were denatured with loading buffer, resolved in SDS/5%polyacrylamide gels and transferred to poly vinylidene difluoride (PVDF)membranes (Millipore Corp., Bedford, Mass.). Membranes were be blockedwith 3% nonfat dry milk in phosphate buffered saline-0.5% Tween 20(PBST) and exposed to primary antibody, anti-smooth muscle alpha-actin(SMA) (Santa Cruz, Ca.) diluted in blocking buffer overnight at 4° C.After incubation, membranes were washed with PBST. After wash, membraneswere exposed to an alkaline phosphatase-conjugated anti-rabbit secondaryantibody for 1 hour. Membranes were subsequently washed with PBST,incubated with chemiluminescence reagents and exposed to x-ray film. ForERK1/2 phosphorylation, VSMC were pre-treated with NRG-1β for 24 hoursand stimulated for 15 minutes with PDGF. Western blots were performedusing primary antibodies for phosphorylated and unphosphorylated formsof ERK1/2 (Cell Signaling, Danvers, Mass.) diluted 1:250 in blockingbuffer. Immunoblotting using an anti-tubulin antibody was used tonormalize protein levels in each sample.

Cell Viability Assay

Quantitative viability assessment was performed using 1% Calcein-AM(Molecular Probes, Eugene, Oreg.), a fluorescent membrane-integrity dye,diluted in HBSS according to the manufacturer's protocol. Qualitativeassessment of cell viability in treated cells was performed using thetrypan blue-exclusion assay. Non-viable cells were quantified visuallyusing a light microscopy.

Statistical Analysis

Each experiment was repeated a minimum of three times. Data areexpressed as the mean±standard deviation (SD). An unpaired Student'st-test and ANOVA were performed to make comparisons between groups. Avalue of p less than 0.05 was considered significant.

Results

NRG-1 Attenuated Neointima Formation after Rat Carotid Balloon Injury

Neointimal hyperplasia was histologically evident in the carotidarteries 14 days after balloon injury compared to uninjuredcontralateral controls. The neointima of the rats receiving intravenousadministration of NRG-1 was significantly reduced compared toballoon-injured animals. Morphometric analysis showed that NRG-1 reducedthe size of the lesion by ˜50% compared to vehicle-treated controlanimals. Treatment of animals with NRG-1 showed no overt negative sideeffects and there was no significant difference in body weight observedamong the control and NRG-1 treated rats.

NRG-1 Inhibits Proliferation in VSMC

One possible mechanism for the inhibitory effect of NRG-1 on neointimalformation is the regulation of pathological VSMC functions. To examinethe effects of NRG-1 on VSMC proliferation, serum-starved VSMC werepre-treated with NRG-1 for 24 hours, then stimulated with PDGF for anadditional 48 hours. Stimulation of cells with PDGF increasedproliferation of VSMC 2-fold. Pre-treatment with either NRG-1β (FIG. 1)or NRG-1α resulted in a dose-dependent decrease in baseline andPDGF-stimulated proliferation as measured by MTS activity. Direct cellcounting using Coulter counter demonstrated that NRG-1 reducedPDGF-stimulated VSMC proliferation, but not baseline cell numbers.Analysis of BrDU incorporation revealed a similar pattern to the Coultercounter demonstrating that NRG-1β significantly inhibited PDGF-inducedproliferation, but did not alter baseline DNA synthesis.

To determine whether the growth inhibitory effects of NRG-1 were due totoxicity or damage to the cells rather than proliferation, calcein-AMand trypan blue viability assays were carried out in cells pre-treatedwith NRG-1 with or without PDGF. The calcein-AM assay demonstrated thattreatment of VSMC with NRG-1 does not alter cell viability. Theseresults were corroborated using the trypan-exclusion assay, whichrevealed that less than 1.0% of the cells took up the dye.

NRG-I Decreases VSMC Migration

The migration of VSMC was measured using a transwell migration assay.VSMC were pretreated with 100 nM NRG-1α or NRG-1β, and then stimulatedwith 10 ng/ml of PDGF-BB for 48 hours. Our results show that NRG-1 alonedoes not alter baseline VSMC migration. VSMC treated with PDGF displayeda 2-3 fold increase in migration. Both NRG-1α and NRG-1β decreasedPDGF-stimulated VSMC migration by 80% and 90%, respectively.

NRG-1 Regulates Smooth Muscle α-Actin Expression

To examine the possibility that NRG-1 may block VSMC proliferation andmigration by preventing de-differentiation, the mRNA and proteinexpression on SMA, a marker for differentiated and contractile VSMC,after NRG-1 treatment was examined. Serum-starved, quiescent VSMCdisplayed SMA expression, which was reduced after treatment with PDGF.NRG-1β alone did not alter SMA mRNA or protein expression, however,pre-treatment of PDGF-stimulated VSMC with NRG-10 resulted in SMAexpression that returned to near baseline levels.

NRG-1 Inhibits PDGF-Induced Phosphorylation of ERK1/2

Several studies have shown that PDGF-induced VSMC proliferation involvesthe ERK, signaling pathway. Regulation of the phosphorylation of thesekinases was used to determine whether NRG-1 could inhibit PDGF activityin VSMC by interfering with ERK activity. PDGF stimulation of VSMCresulted in an induction of ERK1/2 phosphorylation. Treatment with NRG-1alone did not alter ERK1/2 phosphorylation compared to control untreatedVSMC. However, NRG-1 prevented PDGF-induced phosphorylation of ERK1/2.Densitometric revealed that NRG-1 reduced PDGF-stimulated ERK1/2phosphorylation in VSMC by 70%.

While the NRG-1/erbB system had previously been shown to modulatevarious biological activities including cell survival, proliferation,and migration, which are critical for normal development and pathologyin a variety of tissues, the role for NRG-1 in vascular function andinjury has not been clearly elucidated. This study, demonstrated thatNRG-1 attenuates neointimal formation and vascular balloon injury. NRG-1reduced the size of the lesion by ˜50% compared to vehicle-treatedcontrol animals. This novel finding clearly shows that NRG-1 is usefulin the prevention of vascular diseases such as restenosis andatherosclerosis. The NRG-1 blocks PDGF-induced proliferation of VSMC ina dose-dependent manner. The inhibitory effects of NRG-1 on VSMCproliferation were confirmed by direct cell counting and measuring DNAsynthesis by BrDU incorporation. An intriguing observation was thedifference in the effect of NRG-1 on baseline VSMC proliferation usingthe MTS-based assay compared to the other methods. In the cell countingand BrDU approaches, PDGF increased VSMC proliferation was blocked byNRG-1, however, baseline VSMC numbers were not altered. Using theMTS-based assay, a 50% decrease in baseline MTS activity was seen afterNRG-1 administration. Since the MTS assay measures metabolic activity,it is possible that NRG-1 may prevent PDGF-stimulated proliferation bypromoting VSMC differentiation, which could result in a decrease inmetabolic activity and/or a reduction in the capability of PDGF tostimulate VSMC proliferation. That this is due to apoptosis resultingfrom treatment is unlikely since there was no evidence of increased deador non-viable cells after neuregulin treatment.

Combination Therapy to Prevent Permanent Neuronal Damage.

In the case of prevention of damage resulting from exposure toneurotoxins such as organophosphates or as a result of obstructivestroke such as that caused by an infarct studies were done studyingeffect on permanent middle cerebral artery occlusion (pMCAO) usingcombination therapy. Studies were done giving dizocilpin maleate (MK-801from Sigma), a glutamate receptor inhibitor which blocks the excitotoxicevents of ischemia in combination with neuregulin within a therapeuticwindow of about 13.5 hours in the rat to decrease permanent neuronaldamage. The therapeutic window in larger animals having a lowermetabolism would be in the range of 0 to 72 yours. In the case ofexpected exposure to neurotoxins, the neuregulin could be administeredin conjunction with antidotes. Other active agents which may be used inconjunction with neuregulin in the manner disclosed for use with MK-801are selfotel, aptiganel, magnesium, acetylcholine, GABA agonists(clomethiazole, diazepam and other benzodiazepines) and serotoninagonists.

In the case of damage arising from exposure to neurotoxins currentpost-exposure medical countermeasures against nerve agents (e.g.atropine, prostigmine glutamate antagonists, oximes (such as 20pralidoxime chloride) and benzodiazepines) are useful in preventingmortality, but are not sufficiently effective in protecting the CNS fromseizures and permanent injury. Therefore, new and more effective medicalcountermeasures against OP nerve agents are needed to facilitate bettertreatment that will prevent extensive, permanent nerve damage insurvivors. Other agents that may be used to treat patients that havebeen exposed to neurotoxin include anticonvulsants.

In both instances of pMCAO and exposure to neurotoxins, the neuregulinmay be administered concurrently with the other active agents toameliorate permanent damage from infarct disintegrators or nerve agentcounteractants, but should be given within a 72 hour widow after theinitial exposure to the causative agent or the onset of occlusion of theblood supply, more preferably within 24 hours after the causal event.

In both instances where the neuregulin is given as combination therapyto prevent cerebral neuronal damage the neuregulin is administered intothe carotid artery with an appropriate carrier. In animal studies, theneuregulin is administered in bovine serum albumin. In humans, apreferred carrier would be human serum to be administered within thefirst 72 hours, preferably within the first 24 hours, of the assault,whether chemical or physical. (In the instance where the neuregulin isto prevent damage resulting from mechanical damage to a blood vessel,the neuregulin may be given intravenously in the usual carriers used forintravenous administration. Addressing the use of neuregulinsimultaneously with other agents, studies were done on rats that hadbeen subjected to left middle cerebral artery occlusion (MCAO).

Methods

Middle Cerebral Artery Occlusion

All surgical procedures were performed by sterile/aseptic techniques inaccordance with institutional guidelines. Adult male Sprague-Dawley ratsweighing 250-300 g were used for this study. Animals were subjected toleft MCA occlusion. Rats were anesthetized with a ketamine/xylazinesolution (10 mg/kg, IP). MCA occlusion was induced by the intraluminalsuture MCAO method as previously described (Belayev et al. 1996; Belayevet al. 1995). Briefly, the left common carotid artery (CCA) was exposedthrough a midline incision and was carefully dissected free fromsurrounding nerves and fascia. The occipital artery branches of theexternal carotid artery (ECA) were then isolated, and the occipitalartery and superior thyroid artery branches of the ECA were coagulated.The ECA was dissected further distally. The internal carotid artery(ICA) was isolated and carefully separated from the adjacent vagusnerve, and the pterygopalatine artery was ligated close to its originwith a 6-0 silk suture. Then, a 40 mm 3-0 surgical monofilament nylonsuture (Harvard Apparatus, Holliston, Mass.) was coated withpoly-L-lysine with its tip rounded by heating near a flame. The filamentwas inserted from the external carotid artery (ECA) into the internalcarotid artery (ICA) and then into the circle of Willis to occlude theorigin of the left middle cerebral artery. The suture was inserted 18 to20 mm from the bifurcation of the CCA to occlude the MCA. In thepermanent MCAO (pMCAO), the suture was left in place for 24 hours priorto sacrificing the animal. In the transient MCAO (tMCAO) model, thenylon suture was withdrawn 1.5 hours following ischemia and the braintissues were reperfused for 24 hours before sacrificing. To determinethe effects of NRG-1 on ischemic stroke, rat were injectedintra-arterially with a single bolus 10 μl dose of vehicle (1% BSA inPBS) or NRG-1β (10 nmol/L, NRG-1 (EGF-like domain, R&D Systems,Minneapolis, Minn.) in 1% BSA in PBS) through a Hamilton syringe. Thisresulted in the administration of ˜2.5 ng of NRG-1/kg body weight NRG-1or vehicle was administered by bolus injection into the ICA through ECAimmediately before MCAO. MK-801 (0.5 mg/kg) was either administered IPimmediately prior to NRG-1 administration or co-administered IAsimultaneously with NRG-1. All NRG-1 and vehicle treatment studies wereperformed-in a double-blinded manner. Core body temperature wasmonitored with a rectal probe and maintained at 37° C. with aHomeothermic Blanket Control Unit (Harvard Apparatus) during anesthesia.Neurological score was determined in a double blinded fashion using afive-point neurological evaluation scale (Menzies et al. 1992) in ratstreated with vehicle or NRG-1 four hours after reperfusion. All animalswere tested prior to surgery (controls) and after treatment with NRG-1or vehicle. Neurological function was graded on a scale of 0-4 (normalscore 0, maximal deficit score 4). While intra-arterial injection intothe carotid artery was used, fluoroscopic guided catheter-based therapywherein the catheter is guided to the arteries which best access thedamaged tissue is appropriate.

Measurement of Infarct Formation

Twenty-four hours after reperfusion, the animals were killed and thebrain tissue was removed and sliced into 2.0 mm-thick sections. Brainslices were incubated in a 2% triphenyltetrazolium chloride (TTC)solution for 30 minutes at 3° C. and then transferred into a 4%formaldehyde solution for fixation. TTC, a colorless salt, is reduced toform an insoluble red formazan product in the presence of a functioningmitochondrial electron transport chain. Thus, the infarcted region lacksstaining and appears white, whereas the normal non-infarcted tissueappears red. Infarct area of four slices of 2 mm coronal sections ofeach brain was calculated in a blinded manner by capturing the imageswith a digital camera. Rats showing tremor and seizure (which rarelyoccurred in this study) were excluded from studies of brain infarctionto eliminate cerebral hemorrhage or brain trauma as potential variablesin this study. Infarct volumes were analyzed by ANOVA; P<0.05 wasregarded as significant.

While it had previously been demonstrated that a single 2.5 ng/kgintra-arterial administration of NRG-1 prior to MCAO prevented neuronaldeath following ischemia and reperfusion, there was no indication thatuse at or after time of assault, whether mechanical (as with an infact)or chemical, would be effective to ameliorate damage arising from theassault.

Neuregulin also has use, with similar dosage for intravenousadministration in conjunction with reperfusion therapy such asanticoagulant therapy to ameliorate damage to the artery. In suchinstances, the neuregulin may be administered in carriers such asglucose, saline, Ringer's lactate, etc.

Agents with other mechanisms of action that prevent or avoid formationof obstructive occlusion such as those which cause clots to dissolve canbe used with neuregulin. Tissue plasminogen activator (t-PA) can also beused in conjunction with neuregulin. At present, use of t-PA remainslimited and must be administered within three hours of the observedischemic event. However, t-PA patients are at high risk of hemorrhagictransformation. Furthermore, t-PA causes inflammatory responses andreperfusion injury in the brain. The t-PA is administered intravenouslyin saline or similar carriers. In all instances, the neuregulin is mosteffective if administered into the carotid artery in a carriercontaining serum albumin (in the case of humans, human serum albumin).The agents may be administered essentially simultaneously or theneuregulin may be administered within the 0-72 hour time period, thoughit is preferred practice to administer the neuregulin within 24 hours ofadministration of the t-PA.

Intrathecal Use of Neuregulin to Encourage Migration of Stem Cells forProliferation of New Neuronal Cells

While treatments cited above may be effective in limiting damage from apathology-causing event, the recovery of function can take place onlywith regeneration of neuronal tissue. The administration of neuregulininto the ventricular zone provides means for enhancing migration of stemcells which are formed in the ventricle to the site of neuronal damage

The Effect of NRG-1 on NSCs Isolated from E11 Mouse Telencephalon

To investigate the effects of NRG-1 in multipotent NSCs, thetelencephalon of E11 mouse embryos were isolated and the dissociatedcells cultured as neurosphere cultures. In the present study, cultureswere treated with the EGF-like domain of neuregulin-1β (NRG-1). TheEGF-like domain contains the receptor binding portion of the moleculeand has been shown to display all the known biological activities of thefull-length neuregulins. The cells formed neurospheres and expressednestin, an intermediate filament protein present in NSCs and RPs in thedeveloping CNS. The cultures were examined to determine whether theaddition of NRG-1 to cell suspensions obtained from E11 mouse corticaltissue would generate neurospheres in the absence of bFGF. After 7 daysin culture, there was no significant difference in the total number orsize of neurospheres in the NRG-1 treated group compared with theuntreated group, This result demonstrated that NRG-1 alone, unlike bFGF,could not generate neurospheres. When bFGF-generated neurospheres wereplated onto coated coverslips in the presence of bFGF, cells continuedto divide and migrate out of the sphere to form a monolayer. Uponwithdrawal of bFGF, migrating cells differentiated into cells expressingneuronal, astrocyte and oligodendrocyte markers. Neuronal cells wereidentified by labeling with the anti-MAP2 antibody.

Oligodendrocytes were identified with an antibody directed against 04and astrocytes were identified with an antibody directed against GFAP.Morphologically, these MAP2-positive cells appeared neuronal and showed:(i) a spherical, ovoid, or pyramidal shaped soma; (ii) phase-brightappearance; (iii) branching processes (presumably dendrites) arisingfrom the soma.

NRG-1 Increases the Proliferation of MAP2-Positive Cells in NeurosphereCulture.

The actions of NRG-1 on bFGF-generated neurospheres were examined byplating neurospheres on coverslips as described above. Neurospheres werecultured in the absence or presence of 5 nM NRG-1 for 5 days, and thenco-labeled with BrDU and MAP2 or GFAP antibodies. After 5 days oftreatment with 5 nM NRG-1, a dramatic increase in the number of cellssurrounding the core of the neurosphere was observed in NRG-1 treatedcultures as compared to control. A 44±3.3% increase in [³H]thymidineincorporation was seen in NRG-1 treated cultures that paralleled theincrease in the total number of cells. More MAP2 and BrDU co-labeledcells (yellow) were found both in the central core and peripheral areaof NRG-1 treated neurospheres, but few double-labeled cells were seen inthe control. There was a 2.5-fold increase in MAP2 positive cells, butno increase in MAP2-negative cells, suggesting that the majority ofNRG-1 treated cultures were neuronal.

To further characterize the effect of NRG-1 on NSCs, neurospheres werecultured in the absence or presence of 1 or 5 nM NRG-1, then co-labeledwith BrDU and MAP2 or GFAP antibodies. After 5 days, there was a 4-foldincrease in the number BrDU-labeled cells in the neurosphere outgrowtharea in 5 nM NRG-1 treated group compared to control. A smaller, butsignificant increase was also observed with 1 nM NRG-1 treatmentdemonstrating a dose-dependent response of cells to NRG-1. Most of theBrDU positive cells co-labeled with the MAP2, but not the GFAP antibody.Therefore, the increased proliferation was specific for neuronal cellsand not in GFAP-positive astrocytes. The increase in number of MAP2positive cells that co-labeled with BrDU was parallel to the increase ofBrDU positive cells, suggesting that most of the cells proliferating inresponse to NRG-1 were neuronal. In cultures maintained for 8 days afterwithdrawal of bFGF, virtually no cells showed BrDU incorporation incontrol cultures. By that time point, most cells had differentiated andlost the ability to proliferate. However, numerous BrDU-positive neuronswere present in the NRG-1 treated group, suggesting that NRG-1 prolongsthe proliferation of immature neuronal ceils (data not shown). Under ourculture conditions, few cells were labeled with GFAP and O₄ in controlculture or after treatment with NRG-1, therefore the cells that labeledwith BrDU alone were likely undifferentiated NSCs.

NRG-1 Increases Proliferation Rather than Survival.

The increased production of neurons could be altered by affecting (1)the proliferation of NRPs (2) the differentiation of NSCs into neurons,or (3) or by altering the survival of neuronal cells. Increasedproliferation might result in an increase in the total number of cellsas well as in the number of proliferating MAP2-positive cells; increaseddifferentiation might result in an increase in the number ofMAP2-positive cells within the same total population of cells; increasedsurvival might result in an increase in MAP2-positive cells, but notnecessarily cells co-labeled with BrDU or nestin. To determine whetherthe increase in the number of NRPs induced by NRG-1 was due to theincrease of cell survival, we evaluated cell viability by using aViability Assay Kit (Molecular probes). Results showed that the totalnumber of cells increased after 4 days in the cultures. The number oflive cells was greater in NRG-1 treated group compared to control. Twiceas many live cells were present in the NRG-1 group after 8 days. Therewas no difference in the number of dead cells in control or NRG-1treated cultures at most time points. This result shows that NRG-1stimulated proliferation rather than cell survival.

NRG-1 Stimulates the Mobilization of NSCs in Adult Rat Brain In Vivo.

To examine whether NRG-1 could stimulate neurogenesis in vivo, welabeled SVZ cells by stereotaxically injecting DiI into the lateralventricle. Twenty-four hours later, NRG-1 or vehicle were injected intothe lateral ventricle and sacrificed animals 1 day later. Intenselabeling was visible in the cells lining the ventricle (V) and in thechoroids plexus (CP) after injection of DiI. When the vehicle wasinjected 24 after the DiI labeling, few cells had migrated out from theSVZ. However, after NRG-1 administration, numerous NSCs had migratedfrom the SVZ, as far away as to the cerebral cortex. Similar resultswere seen when Fluorogold was injected into the lateral ventricle.Preliminary results indicate that a subpopulation of the labeled cellsco-label with an antibody for NeuN, a neuronal marker (not shown).

The poor regenerative capacity of the sensory of the mammalian CNS hasled to investigations of different approaches to increase the functionof these structures after neurodegeneration or injury. One strategy torepair the injured CNS has been to replace the lost neurons withembryonic stem cell-derived neuronal stem cells (eNSCs). The use ofeNSCs has shown promise in the treatment of a variety of neurologicaldiseases and they have recently been shown to survive and differentiateinto glia and neurons after CNS transplantation. However, a number ofbiological and ethical issues have slowed this area of research. NSCshave been demonstrated in the adult brain and have been shown to havethe potential to differentiate into a variety of neuronal cell types.Therefore, another strategy has focused on maximizing the potential ofthis endogenous population of cells by stimulating their mobilization,proliferation, migration, and differentiation in vivo following CNSinjury and degeneration. Understanding the technical and logisticconsiderations for employing adult NSCs is essential to optimizing andmaintaining cell survival before and after activation, as well as fortracking the fate of mobilized cells. It is now recognized that NSCstrategies will be effective only if the new cells have the sameabilities and characteristics as the original neurons. Before the fullpotential of adult NSCs can be recognized for treating CNS injury, wemust to identify the sources of stem cells, understand factors that canregulate their proliferation, fate specification, and, most importantly,to characterize their functional properties.

The administration of NRG-1 into the cerebral spinal fluid or through ashunt into the ventricle for repeated administration (intrathecaladministration) gives a method of encouraging production of stem cellsin the ventricle with migration of stem cells from the ventricle to thedamaged areas of the brain. Administration may be used with spinal tapor may be administered through a shunt into the ventricle. Appropriatecarriers include glucose, isotonic glucose and other carriers usuallyused for intrathecal administration. Dosage will vary with the size andcondition of the animal, with range of 0.005 to 3 ng/Kg, with dosage ofabout 0.005 to 0.5 ng/Kg being administered to larger mammals. However,the neuregulin may be administered into the cerebral spinal fluid at thelumbar region. Dosage compositions would contain 0.05 to 100 ng in apharmaceutically acceptable carrier. For arterial administration, thecarrier may, advantageously, contain serum albumin.

When administered in conjunction with another active agent such as anantidote to a neurotoxin, an agent to dissolve clots or interfere withclot formation or some other active agent, such agents will be given inthe manner usual for administration of such agent, often intravenously.However, to obtain maximum benefit, the neuregulin will usuallyadminister into the carotid artery or by some other means such asfluoroscopy guided catheter-based means that will provide arterialaccess to the brain.

The use of shunts into the ventricle is well established practice in themedical community. Such shunts may be present for several day or weeks.While usually used to drain excess cerebral spinal fluid from theventricle in cases of excessive production or blockage of flow, suchshunts would be appropriate means for administration of neuregulin overa period of several weeks. The care of the shunt would be an ongoingresponsibility of the medical team during the time neuregulin is beingadministered to facilitate migration of the stem cells to areas ofdamage.

For purposes of regeneration of neuronal tissue, the administration ofNRG-1 should commence after the initial inflammation due to the assaulthas subsided. Because it is necessary for the stem cells that havemigrated to be replenished in the ventricle, the intrathecaladministration of neuregulin should not be repeated at less than oneweek intervals. Longer intervals may be appropriate in order to allowgreater replenishment of the stem cell supply in the ventricle.

While NRG-1 has been exemplified, neuregulins 2, 3, and 4 have beenshown to have similar activity and would also be appropriate for usestaught herein.

1. A method of ameliorating the neuronal damage caused by anorganophosphate neurotoxin, comprising: administering to a subject inneed of such treatment a composition containing 0.05 to 100 ng ofneuregulin-1, wherein said neuregulin-1 is administered intravenously,intra-arterially, or intrathecally.
 2. The method of claim 1, whereinsaid neuregulin-1 is administered in conjunction with tissue plasminogenactivator (t-PA).
 3. The method of claim 1, wherein said neuregutin-1 isadministered in conjunction with serum albumin.
 4. The method of claim1, wherein said neuregulin-1 is administered in conjunction with anantidote to the neurotoxin.
 5. The method of claim 1, wherein saidneuregulin-1 is administered in conjunction with an anticonvulsant. 6.The method of claim 1, wherein said neuregulin-1 is administered inconjunction with an agent selected from the group consisting ofselfotel, aptiganel, magnesium, acetylcholine, GABA agonists andserotonin agonists.
 7. A method of ameliorating neuronal damage arisingfrom exposure to an organophosphate neurotoxin, comprising:administering to a mammal that has been exposed to said neurotoxin of aneuronal damage inhibiting amount of neuregulin-1 wherein theneuregulin-1 is administered within 72 hours of exposure to saidneurotoxin, wherein said neuregulin-1 is administered intravenously,intra-arterially, or intrathecally.
 8. The method of claim 7 wherein thedosage of neuregulin-1 administered to is 0.05 to 5 ng/kg.
 9. The methodof claim 7, wherein said neuregulin-1 is administered in conjunctionwith tissue plasminogen activator (t-PA).
 10. The method of claim 7,wherein said neuregulin-1 is administered in conjunction with serumalbumin.
 11. The method of claim 7, wherein said neuregulin-1 isadministered in conjunction with an antidote to the neurotoxin.
 12. Themethod of claim 7, wherein said neuregulin-1 is administered inconjunction with an anticonvulsant.
 13. The method of claim 7, whereinsaid neuregulin-1 is administered in conjunction with an agent selectedfrom the group consisting of selfotel, aptiganel, magnesium,acetylcholine, GABA agonists and serotonin agonists.
 14. A method ofameliorating damage in central nerve system (CNS) caused by anorganophosphate neurotoxin, comprising: administering to a subject inneed of such treatment a composition containing 0.05 to 100 ng ofneuregulin-1.
 15. The method of claim 14, wherein said neuregulin-1 isadministered in conjunction with tissue plasminogen activator (t-PA), anantidote to the neurotoxin or an anticonvulsant.
 16. The method of claim14, wherein said neuregulin-1 is administered intravenously,intra-arterially, or intrathecally.
 17. The method of claim 14, whereinsaid neuregulin-1 is administered in conjunction with an agent selectedfrom the group consisting of selfotel, aptiganel, magnesium,acetylcholine, GABA agonists and serotonin agonists.